| Antioxidant peptides were prepared from the protein of large yellow croaker (Pseudosciaena crocea) viscera by Alcalase with controlled enzymatic hydrolysis. The antioxidant peptides of Pseudosciaena crocea viscera were fractionated by several ultrafiltration membrane, and the fraction with the best antioxidant activity was chosed for following experiments. The oxidative damage of HepG2 as model of osteobla was induced by hydrogen peroxide, and the influence of the survival rates and the stability of antioxidant defense system in oxidative damage HepG2 with Pseudosciaena crocea viscera peptides pre-treatment was studied, and the suppression effect of inflammation induced by ROS was also discussed. In addition, in order to provide a technical basis for its application, the basic physical,functional properties and oxidative stability of Pseudosciaena crocea viscera peptides were also studied.The basic composition of large yellow croaker viscera was analyzed, and amino acid composition was also detected to evaluate the nutritive value. The content of weight protein, moisture, fat and ash of large yellow croaker were 76.05%,15.68%,1.39%and 4.54%, respectively. The proportion of essential amino acid content was 42.15%, most of which were higher than ideal protein pattern except for valine. Among which the proton affinity amino acids, aromatic amino acids and hydrophobic amino acids accounted for 46.28%,11.30% and 41.22% respectively. It indicated the amino acid composition was reasonable, which showed a good protein resource for antioxidant peptides preparation.Antioxidant peptides were enzymatically prepared from Pseudosciaena crocea viscera protein using Alcalase protease. The optimization was performed using single factor method and response surface methodology based on degree of hydrolysis, DPPH · and hydroxy scavenging ability. The optimal hydrolysis conditions were determined as hydrolysis temperature of 62 ℃, pH of 9, enzyme concentration of 4.26%, substrate concentration of 8 g/100 mL and hydrolysis time of 3.7 h. Under these conditions, the scavenging activity of DPPH · and · OH were up to 85.97% and 75.79% respectively. Moreover, the peptides were isolated by serval ultrafiltration membrane with molecular weight cut-off of 10 kDa,5 kDa and 3 kDa respectively. Four different molecular weight range of peptides were got as followed:I (10 kDa), Ⅱ (5-10 kDa), III (3-5 kDa) and IV (< 3 kDa). IV (< 3 kDa) exhibited the best antioxidant activity compared with other fractions. Therefore, IV was chosed to study in following eaperiments.Pseudosciaena crocea viscera peptide exerted good antioxidant activities in all evaluation system in vitro. The oxidative damage of HepG2 as model of osteobla was induced by hydrogen peroxide. Pseudosciaena crocea viscera peptide pre-treament significantly reduced H2O2 induced oxidative damage in HepG2 cells, increased the cell viability, and significantly increased the levels of SOD, CAT, GSH-Px and GSH in the cells, and recovered the stability of antioxidant defense system. Additionally, the inflammatory factor levels induced by ROS were suppressed significantly. Therefore, Pseudosciaena crocea viscera peptide showed a good antioxidant and anti-inflammatory activity.The physicochemica and functional properties of Pseudosciaena crocea viscera peptide under different conditions and different concentrations were studied, which include solubility, water absorption capacity, water holding capacity, oil absorption capacity, apparent viscosity, foaming properties and emulsifying properties. The results showed the PI of Pseudosciaena crocea viscera peptide was about pH6, at which emulsibilityand foam stability reached the lowest and then had a rapid rising trend. However, emulsion stability, foamability were increased firstly with the pH increasing and then decreased, which met the bottom at pH6.The increase of peptide concentration possessed higher viscosity, emulsion stability and foam stability. On the contrary, the solubility, emulsibility got significant reduction while peptide concentration increased. In addition, foam stability did not have a significant changed as peptide concentration increased. Moreover, compared with traditional emulsifier Tween 20,10% Pseudosciaena crocea viscera peptide were not only decreased MDA in emulsifier about 84.5% after 150 min, but also showed inhibition of flocculation.The effects of NaCl, temperature, pH, ultraviolet radiation, sugars, metal ions, and simulated gastrointestinal digestion were assessed on the antioxidant activity of Pseudosciaena crocea viscera peptides. Under conditions of neutral pH, room temperature, low NaCl concentration, and appropriate amounts of K+,Ca2+, and Al3+, the antioxidant activity ofthe antioxidant peptides from Pseudosciaena crocea viscera was generally stable, and DPPH·radical scavenging rate and Fe2+ chelatingcapacity were 88.5% and 66.7%, respectively. With high NaCl concentration, high temperature, additions of K+, Ca2+, and Al3+, andlong-duration ultraviolet irradiation, the antioxidant activity of the antioxidant peptides from Pseudosciaena crocea viscera was reduced by 30l. Furthermore, at acidic pH and additions of glucose, fructose, and lactose at 70 ℃, the DPPH-radical scavenging activity of the antioxidantpeptides from Pseudosciaena crocea viscera was increased to approximately 90%; while alkaline conditions were conductive for the Fe2+chelating capacity, indicating that treatments have different impacts on different antioxidant indicators. In addition, pepsin digestion treatment had no significant impact on the antioxidant activity of antioxidant peptides from Pseudosciaena crocea viscera, and further chymotrypsin digestion significantly decreased the antioxidant activity stability of the peptides. Therefore, appropriate processing and storage are critical for maintenance of the stability of the antioxidant peptides extracted from Pseudosciaena crocea viscera.Pseudosciaena crocea viscera peptide with Mw<3kDa was isolated and purified by chromatographic methods including ion-exchange chromatography, gel chromatography and reverse high-performance liquid chromatography (RP-HPLC). The results showed the acidic fractions with positively charged, low molecular weight and high hydrophobic in Pseudosciaena crocea viscera peptide exerted better antioxidant activity. The antioxidant activity.of purified fraction (the IC50 value of scavenging DPPH radical is 0.72+0.017mg/mL) was significantly higher than other fractions. |
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